African Journal of Plant Science Vol. 2 (5), pp. 034-037, May, 2008 Available online at http://www.academicjournals.org/AJPS ISSN 1996-0824 © 2008 Academic Journals
Full Length Research Paper
Changes in fatty acid composition of polar lipids associated with growth and senescence in leaves of Catharanthus roseus Sanjay Mishra1* and Rajender Singh Sangwan2 1
Department of Biotechnology, College of Engineering and Technology, IFTM Campus, Lodhipur Rajput, Moradabad 244 001, U.P., India. 2 Division of Plant Physiology and Biochemistry, Central Institute of Medicinal and Aromatic Plants (CIMAP), P.O. CIMAP, Lucknow 226 015, India. Accepted 5 May, 2008
Polar lipids are important membrane components of plant cells. They are known to affect certain membrane properties like permeability, fluidity and active transport. In the present study, individual leaf buds were tagged on the day of appearance and sampling began 7 days later. Additional samples were taken at 7 days intervals until leaf abscission occurred about 77 days after tagging. The experiments were performed from July to October, a time period characterized by a day length of 12 ± 1.5 h and average day and night temperature of approximately 30 and 20°C, respectively. The fatty acid composition of polar lipids from young, mature and senescent leaves of Catharanthus roseus was studied in the present study. Green leaves were observed to contain a considerable amount of hexadecatrienoic acid (16:3) in monogalactosyldiglyceride (MGDG), which suggests that C. roseus belongs to 16:3 plants. Further, the percentage of linolenic acid (18:3) in the chloroplast lipids was lower in senescent leaves than that of green tissues. Senescent leaves also had lower proportions of MGDG 16:3 and phosphatidyl glycerol (PG) hexadecanoic acid (16:1). Such selective catabolism of molecular species of these lipids may be suggestive of significant alterations in ultra structure of chloroplast membranes, thus probably affecting the accumulation of indole alkaloids in the leaf tissue. Besides, the age dependent alterations in the fatty acid composition of other polar lipids, namely, phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS) indicate the possibility of degradation of the organelles other than chloroplast (principally vacuoles) in the leaf cells. Key words: Catharanthus roseus, fatty acid composition, glycolipids, leaf development, polar lipids, phospholipids, senescence, sterol. INTRODUCTION Catharanthus roseus (L.) G.Don, known as the common or Madagascar periwinkle, is a perennial and evergreen herb of the dogbane family (Apocynaceae) that was originally native to the island of Madagascar. It has been widely cultivated for hundreds of years and can now be found growing wild in most warm regions of the world, including the Southern U.S. The plants grow one or two
*Corresponding author. E-mail: sanjay_mishra23@rediffmail. com;
[email protected].
feet high, have glossy, dark green leaves (1-2 inches long) and flowers throughout summer. Vincristine (Oncovin) and vinblastine (Navelbine), the main indole alkaloids produced by this plant, are medicinally important, having long been used as anticancerous agents (Blasko and Cordell, 1990; Verpoorte et al., 1991). In fact, these secondary metabolites are produced at extremely low levels within the plants and remain resistant to feasible chemical synthesis due to their complex structures (Hughes and Shanks, 2002; Verpoorte et al., 1999). During last decade, a lot of efforts have been made in view of exploring primary to secondary metabo-
Mishra and Sangwan
lite networks for terpenoid indole alkaloid biosynthesis in C. roseus (Ayora-Talavera et al., 2002; Canel et al., 1998; Hughes et al., 2002; Hughes et al., 2004; van der Fits and Memelink, 2000; Sangwan et al., 1998; Whitmer et al., 2002a, b). However, the lipid metabolism viz-a-viz. qualitative and quantitative production and sequestration of medicinally and biotechnologically valuable phytoche-micals in C. roseus is still a thrust area to work in. Recently, Mishra et al. (2006) have taken initiatives to work on this aspect and led to a hypothesis that the spatial alterations in lipid profiles may be suggestive of concomitant changes in membrane ultrastructure and functions, putatively leading to perturbation in indole alkaloid sequestration capability of the tissues of a species of pharmaceutical significance. Further, polar lipids are important membrane constituents of plant cells. They play a pivotal role by participating in membrane properties like fluidity, permeability and active transport. In fact, any change in physiological state as well as biotic and abiotic factors may influence the chloroplast biochemistry in terms of state and status of polar lipids (Sreenivasulu et al., 1977; Powles, 1984; Sharma and Sanwal, 1992; Mishra and Sangwan, 1998; Mishra et al., 1998). Age-dependent changes in individual polar lipids have been reported for green leaves of cucumber (Ferguson and Simon, 1973) and bean (Fong and Heath, 1977). However, the information on this class of lipids in alkaloid producing plant is very sparse. Koiwai and Kisaki (1979) have reported that glycolipids are degraded more rapidly than phospholipids during flue-cring of tobacco leaves. Later on, Koiwai et al. (1981) studied certain changes in total and polar lipids and their fatty acid composition in tobacco leaves during growth and senescence. To our knowledge, there is no report so far concerning the establishment of putative inter-relationship between lipid status in chloroplast and/or vacuolar membrane and level of alkaloid accumulation in plants. Our earlier studies on Duboisia myoporoides R. Br. indicated significant changes in accumulation of the tro-pane phytochemicals accompanying its various develop-mental phases (Mishra and Sangwan, 1996). Further, we have explained this phasic pattern (Mishra et al., 1998) in view of the essence of a defined cellular organization to facilitate and maintain the accumulation or sequestration of the secondary phytochemicals. Consequently, the temporal trend of various lipid classes and components deserve to be analyzed in this perspective. This paper deals with the age-dependent changes in fatty acid com-position of polar lipids of C. roseus during leaf growth and senescence. MATERIAL AND METHODS Plant materials Leaf samples were harvested from plants of C. roseus growing at
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the experimental farm of the CIMAP, Lucknow (26.5° N latitude, 80.5° E longitude, 120 m altitude, and subtropical zone), India. Individual leaf buds were tagged on the day of appearance and sampling began 7 days later. Additional samples were taken at 7 days interval until leaf abscission occurred about 77 days after tagging. The experiments were performed from July to October, a time period characterized by a day length of 12 ± 1.5 h and average day and night temperature of approximately 30 and 20°C, respectively. Lipid extraction All solvents were glass distilled prior to use. Total lipids were extracted from powdered leaf samples with the aid of CHCl3-MeOH (2:1, v/v) according to the method employed by Mishra and Sangwan (1998) and purification of lipid fraction was performed with help of 0.9% (w/v) NaCl according to Folch et al. (1957). The purified lipid fraction in chloroform was stored under nitrogen –20°C till further use. Separation and identification of polar lipids Polar lipids were separated from the total lipid extract employing 2D- TLC according to Mishra and Sanwal (1994). The plate was developed with chloroform-methanol-aqueous (28%) ammonia (65:35:5, v/v) in first direction followed by chloroform–acetonemethanol-acetic acid-water (5:2:1:1:0.5, v/v) in the second direction. Lipid spots were located under UV light after spraying with Rhodamine 6 G solution. Each spot was identified by chromatography with standards and by spraying with specific reagents for the lipid class (Mishra and Sangwan, 1998). Preparation of fatty acid methyl esters and gas liquid chromatography Each florescent lipid area on the TLC was properly scrapped from the plate and transferred to a test tube containing 5 ml of 5% (v/v) sulphuric acid in methanol. Methanolysis was performed at 40°C overnight. The fatty acid methyl esters (FAME) were extracted with n-hexane, separated and detected using an AIMIL-Nucan gas chromatograph fitted with a stainless steel column (1.8 m x 2 mm, i.d.) of 20% di-ethylene glycol succinate (DEGS) on Chromosorb W (100-120 mesh). The operating conditions were as follows: oven temperature, 190oC; flame ionization detector and injector temperature, 220oC; flow rate of H2 and N2, 30 ml min-1. Employing authentic reference standards performed the identification of FAME. The peak area was calculated by measuring the height multiplied by the width of the peak at half peak height. The values for each fatty acid are given as percent by weight of total fatty acids according to Mishra and Sanwal (1994). Values were statistically calculated as mean ± SD of three independent sets of experiments with triplicates in each set and expressed as mol % of total fatty acids of each polar lipid.
RESULTS AND DISCUSSION The cellular and metabolic regulations and interactions may have direct bearing on the transport and accumulation of indole alkaloids (Table 1) in C. roseus leaf. The amount of polar lipids varied grossly in the same fashion as total lipids (Mishra et al., 2006) except that during maturation and senescence initiation the polar lipids declined to a greater extent than the neutral lipids.
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Table 1. Comparative account of fatty acid composition of each polar lipid among young mature and senescent leaves of Catharanthus roseus.
Lipid MGDG
DGDG
SQDG
PG
PC
PI
PE
PS
Leaf age
Fatty acid composition (mol %) 16:03 18:00 18:01
16:00
16:01
18:02
18:03
Young
4.0±0.40
ND
12.1±.1.13
ND
3.0±0.29
1.9±0.10
79.0±3.23
Mature
2.0±0.10
ND
16.8±1.42
ND
ND
2.1±0.20
79.1±2.39
Senescent
12.1±1.02
ND
4.9±0.79
4.0±0.50
1.1±0.10
4.8±0.30
73.1±2.10
Young
17.0±1.15
ND
ND
2.0±0.21
1.0±0.10
1.0±0.10
79.0±2.32
Mature
15.2±1.09
ND
ND
1.0±0.10
1.9±0.20
1.9±0.20
80.0±2.52
Senescent
25.9±1.93
ND
ND
12.0±1.03
7.0±0.39
3.0±0.2
52.1±1.95
Young
46.0±2.24
ND
ND
4.0±0.42
3.0±0.15
5.0±0.42
42.0±2.02
Mature
56.3±2.39
ND
ND
4.0±0.41
2.0±0.10
3.0±0.23
34.7±0.93
Senescent
48.1±2.10
ND
ND
7.0±0.71
4.0±0.41
5.9±0.45
35.0±0.91
Young
16.0±1.21
33.0±1.28
ND
5.0±0.45
6.0±0.85
9.0±0.76
31.0±0.89
Mature
22.1±.1.13
31.0±1.26
ND
3.0±0.21
4.0±0.40
11.0±1.05
28.9±0.79
Senescent
52.0±1.82
6.0±0.91
ND
12.0±1.05
6.0±0.88
9.0±0.82
15.0±1.01
Young
33.2±1.16
ND
ND
7.9±0.92
7.0±0.93
30.0±1.23
21.9±1.07
Mature
28.0±1.06
ND
ND
5.0±0.43
6.0±0.88
21.0±1.15
40.0±1.75
Senescent
39.1±1.43
ND
ND
8.0±0.96
7.0±0.90
19.0±1.02
26.9±1.02
Young
25.0±1.20
ND
ND
7.0±0.65
6.0±0.88
32.0±1.16
30.0±1.23
Mature
28.3±1.01
ND
ND
3.9±0.34
7.9±0.89
23.0±1.34
36.9±1.31
Senescent
41.0±1.92
ND
ND
7.0±0.74
8.0±0.89
25.0±1.02
19.0±1.03
Young
21.4±1.81
ND
ND
6.9±0.63
3.9±0.20
37.9±1.29
29.9±1.21
Mature
32.0±1.28
ND
ND
5.0±0.45
6.0±0.88
30.0±1.21
27.0±1.19
Senescent
37.7±1.08
ND
ND
11.1±1.01
10.1±0.92
16.0±1.02
25.1±1.01
Young
44.0±1.99
ND
ND
8.0±0.89
9.0±0.86
23.0±1.71
16.0±1.04
Mature
39.3±2.02
ND
ND
9.9±0.95
10.9±0.91
19.0±1.52
20.9±1.34
Senescent
47.1±.35
ND
ND
4.0±0.39
5.0±0.32
16.0±1.24
27.9±1.26
Values are mean ± SD of three independent sets of experiments with triplicates in each set and are expressed as mol % of total fatty acids of each polar lipid. Young, 28 DAP (Day after proliferation); Mature, 56 DAP; Senescent 77 DAP. ‘ND’ denotes ‘not detected’.
The period of aging and senescence accompanied a slow but consistent trend of decline in polar lipids. Most of polar lipids in C. roseus leaves alter concomitantly with changes in chlorophyll content during development and senescence (Mishra et al., 2006). This study deals with the alterations in fatty acid profile of polar lipids in young, mature and senescent leaves of C. roseus. Fatty acid composition of each glycolipid and PG differed little between young and mature leaves (Table 1). Increases in 18:3 and decreases in linolenic acid (18:2) in glycolipids and PG during greening of leaves have been reported for many plants (Ohnishi and Yamada, 1980a). MGDG and digalactosyl diglyceride (DGDG) were found to possess a very high proportion of 18:3 in young and mature leaves when compared to other phospholipids. MGDG was characterized by 16:3. This suggests that C. roseus belongs to 16:3 plants (Siebertz and Heinz, 1977; Heinz, 1977; Heinz et al.,
1979; Roughan et al., 1979; Koiwai et al., 1981)) and may possess the prokaryotic pathway. Nevertheless, it cannot absolutely preclude the possibility of the employment of the eukaryotic pathway (Roughan et al., 1980; Koiwai et al., 1981) for the synthesis of 18:3-MGDG via oleic acid (18:1)-PC and 18:2-PC have been reported in 18:3 plants such as Avena (Ohnishi and Yamada, 1980a, b). Sulphoquinovosyl diglyceride (SQDG) and PG had relatively high proportions of 18:3 and palmitic acid (16:0) and the latter was characteristic of 16:1. Generally, the relative amount of 18:3 in these four chloroplast lipids dropped considerably in senescent leaves with concomitant rise in 16:0 and stearic acid (18:0), although the contents of all fatty acids declined significantly. The relative amounts of 16:3 in MGDG and 16:1 in PG also decreased in senescent leaves. In fact, these changes are basically consistent with those reported for cucumber (Ferguson and Simon, 1973).
Mishra and Sangwan
Taken together with our observations and discussion in the previous reports on the medicinal plant lipid profile (Mishra and Sangwan, 1998; Mishra et al., 1998; Mishra et al., 2006), it appears that selective degradation occurring in molecular species of these lipids during senescence might be a manifestation of the changing ultra structure and composition of organellar membranes during the phase. Such changes, particularly those concerning vacuoles etc., in turn, might lead to a perturbation of indole alkaloids in the leaf tissue. Specifically, agedependent changes in fatty acid profile of polar lipids like PC, PI, PE, and PS might be related to degradation of the organelles mainly vacuoles in the leaf cells in C. roseus. ACKNOWLEDGEMENTS An institutional research promotion grant to the Department of Biotechnology, College of Engineering and Technology, Moradabad, U.P., India is duly acknowledged. The authors are grateful to Prof. R.M. Dubey (Managing Director), Prof. A. Srivastava (Director) and Prof. B.N. Basu (Dean, Research) of CET, IFTM, Moradabad, U.P., India for providing the necessary facilities and encouragement. Besides, all the faculty members of the Department of Biotechnology, CET, Moradabad, U.P., India are thanked for valuable discussion. REFERENCES Ayora-Talavera TJ, Chappell E, Lozoya-Gloria F, Loyola-Vargas VM (2002). Overexpression in Catharanthus roseus hairy roots of a truncated hamster 3-hydroxy-3-methylglutaryl-CoA reductase gene. Appl. Biochem. Biotechnol. 97:135- 145. Blasko G, Cordell GA (1990). Indole alkaloids: a review. Alkaloids 37:169. Canel C, Lopes-Cardoso M.I., Whitmer S, van der Fits L, Pasquali G, van der Heijden R, Hoge JH, Verpoorte R (1998). Effects of overproduction of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cellcultures of Catharanthus roseus. Planta 205:4-419. Ferguson CHR, Simon EW (1973). Membrane lipids in senescing green tissues. J Exp. Bot. 24: 307-316. Fong F, Heath RL (1977). Age dependent changes in phospholipids and galactolipids in primary bean leaves (Phaseolus vulgaris). Phytochemistry 16: 215- 217. Heinz E (1977). Enzymatic reaction in galactolipids biosynthesis. In Tevini M, Lichtenthaler HK, eds, Lipids and Lipid Polymers in Higher Plants, Springer Verlag Berlin and New York pp 102-120. Heinz E, Siebertz HP, Linschied M, Joyard J, Douce R (1979). Investigations on the origin of diglyceride diversity in leaf lipids. In Appelqvist LA, Lilgenberg C, eds, Advances in the biochemistery and Physiology of Plant Lipids, Elsevier/North- Holland Biomedical Press, Amsterdam pp. 99-120. Hughes EH, Hong SB, Shanks JV, San KY, Gibson SI (2002). Characterisation of an inducible promoter system in Catharanthus roseus hairy roots. Biotechnol. Prog. 18:1183-1186. Hughes EH, Shanks JV (2002). Metabolic engineering of plants for alkaloid production. Metabol Eng. 4:41-48. Hughes EH, Hong S-B, Gibson SI, Shanks JV, San K-Y (2004). Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6:268-276.
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